The invention relates to monitoring of the post-translational modification of a protein.
The post-translational modification of proteins have been known for over 40 years and since then has become a ubiquitous feature of protein structure. The addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells. Such modifications include, but are not limited to, the addition of a carbohydrate (glycosylation), ADP-ribosyl (ADP ribosylation), fatty acid (prenylation, which includes but is not limited to: myrisoylation and palmitylation), ubiquitin (ubiquitination) and sentrin (sentrinization; a ubiquitination-like protein modification). Additional examples of post-translational modification include methylation, actylation, hydroxylation, iodination and flavin linkage. Many of the identified modifications have important consequences for the activity of those polypeptides so modified.
Currently there are several approaches to analyzing the state of modification of target proteins in vivo:
1. In vivo labelling of cellular substrate pools with radioactive substrate or substrate precursor molecules to result in incorporation of labeled (for example, radiolabeled) moieties (e.g., fatty acyl (including, but not limited to, myristoyl and palmityl) sentrin, methyl, actyl, hydroxyl, iodine, flavin, ubiquitin or ADP-ribosyls), which are added to target proteins. Analysis of modified proteins is typically performed by electrophoresis and autoradiography, with specificity enhanced by immunoprecipitation of proteins of interest prior to electrophoresis.
2. Back-labeling. The enzymatic incorporation of a labeled (including, but not limited to, with a radioactive and fluorescent label) moiety into a protein in vitro to estimate the state of modification in vivo.
3. Detection of alteration in electrophoretic mobility of modified protein compared with unmodified (e.g., glycosylated or ubiquitinated) protein.
4. Thin-layer chromatography of radiolabelled fatty acids extracted from the protein of interest.
5. Partitioning of protein into detergent-rich or detergent-poor layer by phase separation, and the effects of enzyme treatment of the protein of interest on the partitioning between aqueous and detergent-rich environments.
6. The use of cell-membrane-permeable protein-modifying enzyme inhibitors (e.g., Wortmannin, staurosporine) to block modification of target proteins and comparable inhibitors of the enzymes involved in other forms of protein modification (above).
7. Antibody recognition of the modified form of the protein (e.g., using an antibody directed at ubiquitin or carbohydrate epitopes), e.g., by Western blotting, of either 1- or 2-dimensional gels bearing test protein samples.
8. Lectin-protein interaction in Western blot format as an assay of the presence of particular carbohydrate groups (defined by the specificity of the lectin in use).
9. The exploitation of eukaryotic microbial systems to identify mutations in protein-modifying enzymes.
These strategies have certain limitations. Monitoring states of modification by pulse or steady-state labelling is merely a descriptive strategy to show which proteins are modified when samples are separated by gel electrophoresis and visualized by autoradiography. This is unsatisfactory, due to the inability to identify many of the proteins that are modified. A degree of specificity is afforded to this technique if it is combined with immunoprecipitation; however, this is of course limited by the availability of antibodies to target proteins. Moreover, only highly-expressed proteins are readily detectable using this technique, which may fail to identify many low-abundance proteins, which are potentially important regulators of cellular functions.
The use of enzyme inhibitors to block activity is also problematic. For example, very few enzyme inhibitors have adequate specificity to allow for the unequivocal correlation of a given enzyme with a specific modification reaction. Indeed, many inhibitors have a broad inhibitory range. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible.
Finally, yeast (Saccharomyces cervisiae and Schizosaccharomyces pombe) has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein-modifying enzymes have been elucidated. However, these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
A non-limiting example of post-translational modification is provided by the Ras proteins, which are a conserved group of polypeptides located at the plasma membrane which exist in either a GTP-bound active state or in a GDP-bound inactive state. This family of proteins operates in signal transduction pathways that regulate cell growth and differentiation. In higher eukaryotes, Ras is a key regulator that mediates signal transduction from cell surface tyrosine kinase receptors to the nucleus via activation of the MAP kinase cascade. Recent studies have demonstrated that Ras directly binds a serine/threonine kinase, Raf-1, a product of the c-raf-1 proto-oncogene, and that this association leads to stimulation of the activity of Raf-1 to phosphorylate MAP kinase kinase (MEK).
An important post-translational modification is the addition of ubiquitin to selected polypeptides. This provides a key mechanism by which to control the abundance of important regulatory proteins, for example, G1 and mitotic cyclins and the p53 tumor suppressor protein. Ubiquitin is a highly conserved 76-amino-acid cellular polypeptide. The role of ubiquitin in targeting proteins for degradation involves the specific ligation of ubiquitin to the xcex5 group of lysine residues in proteins that are to be degraded or internalized from the plasma membrane. The ubiquitin tag determines the fate of the protein and results in its selective proteolysis. Recently a number of factors have been isolated and shown to be involved in the ubiquitination process.
The initial step in the addition of ubiquitin to a protein is the activation of ubiquitin by the ubiquitin activating enzyme, E1. This is an ATP-dependent step resulting in the formation of a thioester bond between the carboxyl terminal glycine of ubiquitin and the active site cysteine residue of E1. Activated ubiquitin then interacts with a second factor, the E2 protein. A thioester bond forms between the activated glycine residue of ubiquitin and a cysteine residue in a specific E2 protein. The E2 proteins represent a family of closely-related proteins encoded by different genes that confer specificity in the proteolytic process. The ligation of ubiquitin to target proteins is effected by the involvement of a further factor, a ubiquitin ligase, E3, of which a number are known (in yeast, reviewed by Haas and Siepmann, 1997, FASEB J., 11: 1257-1268; in humans, see Honda et al., 1997, FEBS Lett., 420: 25-27). E3 completes the final step of ubiquitination by attaching ubiquitin via the xcex5 amino group on lysine residues in proteins to be targeted for degradation. Moreover, E3 is able to add ubiquitin to ubiquitin molecules already attached to target proteins, thereby resulting in polyubiquitinated proteins that are ultimately degraded by the multi-subunit proteasome.
An example of heterodimer association is described in patent application number W092/00388. It describes an adenosine 3:5 cyclic monophosphate (cAMP) dependent protein kinase which is a four-subunit enzyme being composed of two catalytic polypeptides (C) and two regulatory polypeptides (R). In nature the polypeptides associate in a stoichiometry of R2C2. In the absence of cAMP the R and C subunits associate and the enzyme complex is inactive. In the presence of cAMP the R subunit functions as a ligand for cAMP resulting in dissociation of the complex and the release of active protein kinase. The invention described in WO92/00388 exploits this association by adding fluorochromes to the R and C subunits.
The polypeptides are labeled (or xe2x80x98taggedxe2x80x99) with fluorophores having different excitation/emission wavelengths. The emission from one such fluorophore following excitation effects a second excitation/emission event in the second fluorophore. By monitoring the fluorescence emission of each fluorophore, which reflects the presence or absence of fluorescence energy transfer between the two, it is possible to derive the concentration of cAMP as a function of the level of association between the R and C subunits. Therefore, the natural affinity of the C subunit for the R subunit has been exploited to monitor the concentration of a specific metabolite, namely cAMP.
The prior art teaches that intact, fluorophore-labeled proteins can function as reporter molecules for monitoring the formation of multi-subunit complexes from protein monomers; however, in each case, the technique relies on the natural ability of the protein monomers to associate.
Tsien et al. (WO97/28261) teach that fluorescent proteins having the proper emission and excitation spectra that are brought into physically close proximity with one another can exhibit fluorescence resonance energy transfer (xe2x80x9cFRETxe2x80x9d). The invention of WO97/28261 takes advantage of that discovery to provide tandem fluorescent protein constructs in which two fluorescent protein moieties capable of exhibiting FRET are coupled through a linker to form a tandem construct. In the assays of the Tsien et al. application, protease activity is monitored using FRET to determine the distance between fluorophores controlled by a peptide linker and subsequent hydrolysis thereof. Other applications rely on a change in the intrinsic fluorescence of the protein as in the kinase assays of WO98/06737.
The present invention instead encompasses the use of FRET or other detection procedures to monitor the association of polypeptides, as described herein, which are labeled with fluorescent labels (protein and chemical); in the invention, FRET, fluorescence correlation spectroscopy, fluorescence anisotropy, monomer:excimer fluorescence or other techniques indicate the proximity of two labeled polypeptide binding partners, which labeled partners associate either in the presence or absence of a given post-translational modification to an site which is present in the natural binding domain and, optionally, in the binding partner, but not into the fluorophore, reflecting the modification state of one or both of the binding partners and, consequently, the level of activity of a protein-modifying enzyme. The invention further provides methods which employ non-fluorescent labels including, but not limited to, radioactive labels. In addition, the invention encompasses methods which do not employ detectable labels; such methods include, but are not limited to, the detection of the inhibition or reconstition of enzymatic activity, which inhibition or reconstitution results from modification-dependent binding or dissociation between a natural binding domain and a binding partner therefor.
There is a need in the art for efficient means of monitoring and/or modulating post-translational protein modification. Further, there is a need to develop a technique whereby the addition/removal of a modifying group can be monitored continuously during real time to provide a dynamic assay system that also has the ability to resolve spatial information.
The invention provides natural binding domains, sequences and polypeptides, all as defined below, as well as kits comprising these molecules and assays of enzymatic function in which they are employed as reporter molecules.
One aspect of the invention is an isolated natural binding domain and a binding partner therefor, wherein the isolated natural binding domain includes a site for post-translational modification and binds the binding partner therefor in a manner dependent upon modification of the site.
The invention additionally encompasses a method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated natural binding domain and a binding partner therefor as a result of contacting one or both of the isolated natural binding domain and the binding partner with the enzyme, wherein the isolated natural binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site and wherein detection of binding of the isolated natural binding domain and the binding partner as a result of the contacting is indicative of enzyme activity.
Another aspect of the invention is a method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated natural binding domain from a binding partner therefor as a result of contacting one or both of the isolated natural binding domain and the binding partner with said enzyme, wherein the isolated natural binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site and wherein detection of dissociation of the isolated natural binding domain from the binding partner as a result of the contacting is indicative of enzyme activity.
As used herein, the term xe2x80x9cbinding domainxe2x80x9d in a three-dimensional sense refers to the amino acid residues of a first polypeptide required for modification-dependent binding between the first polypeptide and its binding partner. The amino acids of a xe2x80x9cbinding domainxe2x80x9d may be either contiguous or non-contiguous and may form a binding pocket for modification-dependent binding. A binding domain must include at least 1 amino acid, and may include 2 or more, preferably 4 or more, amino acids which are contiguous or non-contiguous, but are necessary for modification-dependent binding to the binding partner, and may include a full-length protein.
A binding domain which is of use in the invention is a xe2x80x9cnatural binding domainxe2x80x9d (i.e., a binding domain that exhibits modification-dependent binding to a binding partner in nature). A natural binding domain of use in the invention may be isolated or may be present in the context of a larger polypeptide molecule (i.e., one which comprises amino acids other than those of the natural binding domain), which molecule may be either naturally-occurring or recombinant and, in the case of the latter, may comprise either natural or non-natural amino acid sequences outside the binding domain.
As used herein with regard to modification of a polypeptide, the terms xe2x80x9csitexe2x80x9d and xe2x80x9csite sufficient for the addition ofxe2x80x9d refer to an amino acid sequence which is recognized by (i.e., a signal for) a modifying enzyme for the purpose of post-translational modification (i.e., addition or removal of a xe2x80x9cmoietyxe2x80x9d as defined below) of the polypeptide or a portion thereof. A xe2x80x9csitexe2x80x9d additionally refers to the single amino acid which is modified. It is contemplated that a site comprises a small number of amino acids, as few as one but typically from 2 to 10, less often up to 30 amino acids, and further that a site comprises fewer than the total number of amino acids present in the polypeptide.
In an enzymatic assay of the invention, a xe2x80x9csitexe2x80x9d, for post-translational modification may be present on either or both of a natural binding domain and its binding partner. If such sites are present on both the natural binding domain and the binding partner, binding between the natural binding domain and its binding partner may be dependent upon the modification state of either one or both sites. If a single polypeptide chain comprises the natural binding domain and its binding partner (or two natural binding domains), the state of post-translational modification of one or both sites will determine whether binding between the two domains occurs.
As used herein, the term xe2x80x9cmodificationxe2x80x9d or xe2x80x9cpost-translational modificationxe2x80x9d refers to the addition or removal of a chemical xe2x80x9cmoietyxe2x80x9d, as described herein, to/from a site on a polypeptide chain and does not refer to other post-translational events which do not involve addition or removal of such a moiety as described herein, and thus does not include simple cleavage of the reporter molecule polypeptide backbone by hydrolysis of a peptide bond, but does include hydrolysis of an isopeptide bond (e.g., in the removal of ubiquitin).
As used interchangeably herein, the terms xe2x80x9cmoietyxe2x80x9d and xe2x80x9cgroupxe2x80x9d refer to one of the post-translationally added or removed groups referred to herein: i.e., one of a ubiquitin moiety, a glycosyl moiety, a fatty acyl moiety, a sentrin moiety or an ADP-ribosyl moiety. A xe2x80x9cmoietyxe2x80x9d or xe2x80x9cgroupxe2x80x9d as defined herein does not refer to a phosphate.
As used herein, the term xe2x80x9cbinding partnerxe2x80x9d refers to a polypeptide or fragment thereof (a peptide) that binds to a binding domain, sequence or polypeptide, as defined herein, in a manner which is dependent upon the state of modification of a site for post-translational modification which is, at a minimum, present upon the binding domain, sequence or polypeptide; the binding partner itself may, optionally, comprise such a site and binding between the binding domain, fragment or polypeptide with its corresponding binding partner may, optionally, depend upon modification of that site. A binding partner does not necessarily have to contain a site for post-translational modification if such a site is not required to be present on it for modification-dependent association between it and a binding domain, sequence or polypeptide. Binding partners of use in the invention are those which are found in nature and exhibit natural modification-dependent binding to a natural binding domain, sequence or polypeptide of the invention as defined herein. In one embodiment of the invention, a binding partner is shorter (i.e., by at least one N-terminal or C-terminal amino acid) than the natural full-length polypeptide.
As used herein, the term xe2x80x9cassociatesxe2x80x9d or xe2x80x9cbindsxe2x80x9d refers to a natural binding domain as described herein and its binding partner having a binding constant sufficiently strong to allow detection of binding by FRET or other detection means, which are in physical contact with each other and have a dissociation constant (Kd) of about 10 xcexcM or lower. The contact region may include all or parts of the two molecules. Therefore, the terms xe2x80x9csubstantially dissociatedxe2x80x9d and xe2x80x9cdissociatedxe2x80x9d or xe2x80x9csubstantially unboundxe2x80x9d or xe2x80x9cunboundxe2x80x9d refer to the absence or loss of contact between such regions, such that the binding constant is reduced by an amount which produces a discernable change in a signal compared to the bound state, including a total absence or loss of contact, such that the proteins are completely separated, as well as a partial absence or loss of contact, so that the body of the proteins are no longer in close proximity to each other but may still be tethered together or otherwise loosely attached, and thus have a dissociation constant greater than 10 xcexcM (Kd). In many cases, the Kd will be in the mM range. The terms xe2x80x9ccomplexxe2x80x9d, xe2x80x9cdimerxe2x80x9d, xe2x80x9cmultimerxe2x80x9d and xe2x80x9coligomerxe2x80x9d as used herein, refer to the natural binding domain and its binding partner in the associated or bound state. More than one molecule of each of the two or more proteins may be present in a complex, dimer, multimer or oligomer according to the methods of the invention.
As used herein in reference to a natural binding domain or other polypeptide, the term xe2x80x9cisolatedxe2x80x9d refers to a molecule or population of molecules that is substantially pure (i.e., free of contaminating molecules of unlike amino acid sequence).
As used herein in reference to the purity of a molecule or population thereof, the term xe2x80x9csubstantiallyxe2x80x9d refers to that which is at least 50%, preferably 60-75%, more preferably from 80-95% and, most preferably, from 98-100% pure.
xe2x80x9cNaturally-occurringxe2x80x9d as used herein, as applied to a polypeptide or polynucleotide, refers to the fact that the polypeptide or polynucleotide can be found in nature. One such example is a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated form a source in nature.
The term xe2x80x9csyntheticxe2x80x9d, as used herein, is defined as any amino- or nucleic acid sequence which is produced via chemical synthesis.
In an assay of the invention, post-translational modification is reversible, such that a repeating cycles of addition and removal of a modifying moiety may be observed, although such cycles may not occur in a living cell found in nature.
An advantage of assays of the invention is that they may, if desired, be performed in xe2x80x9creal timexe2x80x9d. As used herein in reference to monitoring, measurements or observations in assays of the invention, the term xe2x80x9creal timexe2x80x9d refers to that which is performed contemporaneously with the monitored, measured or observed events and which yields a result of the monitoring, measurement or observation to one who performs it simultaneously, or effectively so, with the occurrence of a monitored, measured or observed event. Thus, a xe2x80x9creal timexe2x80x9d assay or measurement contains not only the measured and quantitated result, such as fluorescence, but expresses this in real time, that is, in hours, minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed the instrumentation capability; further, resolution is also limited by the folding and binding kinetics of polypeptides.
As used herein, the term xe2x80x9cbinding sequencexe2x80x9d refers to that portion of a polypeptide comprising at least 1 amino acid, preferably at least 2, more preferably at least 4, and up to 8, 10, 100 or 1000 contiguous (i.e., covalently linked by peptide bonds) amino acid residues or even as many residues as are comprised by a full-length protein, that are sufficient for modification-dependent binding to a binding partner. A binding sequence may exist on a polypeptide molecule that consists solely of binding sequence amino acid residues or may, instead, be found in the context of a larger polypeptide chain (i.e., one that comprises amino acids other than those of the binding sequence).
As used herein in reference to those binding sequences that are of use in the invention, the term xe2x80x9cnatural binding sequencexe2x80x9d refers to a binding sequence, as defined above, which consists of an amino acid sequence which is found in nature and which is naturally dependent upon the modification state of a site for post-translational modificationfound within it for binding to a binding partner. A xe2x80x9cnatural binding sequencexe2x80x9d may be present either in isolation or in the context of a larger polypeptide molecule, which molecule may be naturally-occurring or recombinant. If present, amino acids outside of the binding sequence may be either natural, i.e., from the same polypeptide sequence from which the fragment is derived, or non-natural, i.e., from another (different) polypeptide, or non-natural (a sequence that is not derived from any known polypeptide). In assays of the invention, a binding sequence and its binding partner may exist either on two different polypeptide chains or on a single polypeptide chain.
As used herein, the term xe2x80x9cbinding polypeptidexe2x80x9d refers to a molecule comprising multiple binding sequences, as defined above, which sequences are derived from a single, naturally-occurring polypeptide molecule and are both necessary and, in combination, sufficient to permit modification-state-dependent binding of the binding polypeptide to its binding partner, as defined above, wherein the sequences of the binding polypeptide are either contiguous or are non-contiguous. As used herein in reference to the component binding sequences of a binding polypeptide, the term xe2x80x9cnon-contiguousxe2x80x9d refers to binding sequences which are linked by intervening naturally-occurring, as defined herein, or non-natural amino acid sequences or other chemical or biological linker molecules such are known in the art. The amino acids of a polypeptide that do not significantly contribute to the modification-state-dependent binding of that polypeptide to its binding partner may be those amino acids which are naturally present and link the binding sequences in a binding polypeptide or they may be derived from a different natural polypeptide or may be wholly non-natural. In assays of the invention, a binding polypeptide and its binding partner (which may, itself, be a binding domain, sequence or polypeptide, as defined herein) may exist on two different polypeptide chains or on a single polypeptide chain.
As used herein, the terms xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cpeptidexe2x80x9d refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. The terms subunit and domain also may refer to polypeptides and peptides having biological function. A peptide useful in the invention will at least have a binding capability, i.e, with respect to binding as- or to a binding partner, and also may have another biological function that is a biological function of a protein or domain from which the peptide sequence is derived. xe2x80x9cPolypeptidexe2x80x9d refers to a naturally-occurring amino acid chain comprising a subset of the amino acids of a full-length protein, wherein the subset comprises at least one fewer amino acid than does the full-length protein, or a xe2x80x9cfragment thereofxe2x80x9d or xe2x80x9cpeptidexe2x80x9d, such as a selected region of the polypeptide that is of interest in a binding assay and for which a binding partner is known or determinable. xe2x80x9cFragment thereofxe2x80x9d thus refers to an amino acid sequence that is a portion of a full-length polypeptide, between about 8 and about 1000 amino acids in length, preferably about 8 to about 300, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length. xe2x80x9cPeptidexe2x80x9d refers to a short amino acid sequence that is 10-40 amino acids long, preferably 10-35 amino acids. Additionally, unnatural amino acids, for example, xcex2-alanine, phenyl glycine and homoarginine may be included. Commonly-encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L-optical isomer. The L-isomers are preferred. In addition, other peptidomimetics are also useful, e.g. in linker sequences of polypeptides of the present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267).
As used herein, the terms xe2x80x9cproteinxe2x80x9d, xe2x80x9csubunitxe2x80x9d and xe2x80x9cdomainxe2x80x9d refer to a linear sequence of amino acids which exhibits biological function. This linear sequence includes full-length amino acid sequences (e.g. those encoded by a full-length gene or polynucleotide), or a portion or fragment thereof, provided the biological function is maintained by that portion or fragment. The terms xe2x80x9csubunitxe2x80x9d and xe2x80x9cdomainxe2x80x9d also may refer to polypeptides and peptides having biological function. A peptide useful in the invention will at least have a binding capability, i.e, with respect to binding as or to a binding partner, and also may have another biological function that is a biological function of a protein or domain from which the peptide sequence is derived.
xe2x80x9cPolynucleotidexe2x80x9d refers to a polymeric form of nucleotides of at least 10 bases in length and up to 1,000 bases or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
It is preferred in an isolated natural binding domain and binding partner therefor that the site comprises a sequence which directs modification by one or more of the following enzymes: a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme E1, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase. The site does not comprise a sequence which directs modification by a protein kinase or phosphatase.
It is additionally preferred that the site permits addition of a chemical moiety which may be: a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acid moiety and a sentrin moiety, and the addition prevents binding of the isolated natural binding domain to the binding partner.
As used herein the term xe2x80x9cprevents bindingxe2x80x9d or xe2x80x9cprevents associationxe2x80x9d refers to the ability of at least one of a ubiquitin moiety, a glycosyl moiety, a fatty acyl moiety, a sentrin moiety or an ADP-ribosyl moiety to inhibit the association, as defined above, of an isolated natural binding domain and a binding partner thereof by at least 10%, preferably by 25-50%, highly preferably by 75-90% and, most preferably, by 95-100% relative the association observed in the absence of such a modification under the same experimental conditions.
According to another preferred embodiment, the site permits addition of a chemical moiety which may be: a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acid moiety and a sentrin moiety, and the addition promotes binding of the isolated natural binding domain to the binding partner.
As used herein, the term xe2x80x9cpromotes bindingxe2x80x9d refers to that which causes an increase in binding of the natural binding domain and its binding partner of at least two-fold, preferably 10- to 20-fold, highly preferably 50- to 100-fold, more preferably from 200- to 1000-fold, and, most preferably, from 200 to 10,000-fold.
Preferably, the site permits removal of a chemical moiety which may be: a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acid moiety and a sentrin moiety, and the removal prevents binding of the isolated natural binding domain to the binding partner.
It is preferred that the site permits removal of a chemical moiety which may be: a ubiquitin moiety, a glycosyl moiety, an ADP-ribosyl moiety, a fatty acid moiety and a sentrin moiety, and the removal promotes binding of the isolated natural binding domain to the binding partner.
Preferably, at least one of the isolated natural binding domain and the binding partner comprises a detectable label, more preferably, the detectable label emits light and, most preferably, the light is fluorescent.
A xe2x80x9cfluorescent tagxe2x80x9d, xe2x80x9cfluorescent labelxe2x80x9d or xe2x80x9cfluorescent groupxe2x80x9d refers to either a fluorophore or a fluorescent protein or fluorescent fragment thereof. xe2x80x9cFluorescent proteinxe2x80x9d refers to any protein which fluoresces when excited with appropriate electromagnetic radiation. This includes proteins whose amino acid sequences are either natural or engineered. A xe2x80x9cfluorescent proteinxe2x80x9d is a full-length fluorescent protein or fluorescent fragment thereof. By the same token, the term xe2x80x9clinkerxe2x80x9d refers to the radical of a molecular linker that is coupled to both the donor and acceptor protein molecules, such as an amino acid sequence joining two natural binding domains, sequences or polypeptides or joining a natural binding domain, sequence or polypeptide and its corresponding binding partner, or a disulfide bond between two polypeptide sequences, whether the sequences are present on the same- or on different polypeptide chains.
It is contemplated that with regard to fluorescent labels employed in FRET, the reporter labels are chosen such that the emission wavelength spectrum of one (the xe2x80x9cdonorxe2x80x9d) is within the excitation wavelength spectrum of the other (the xe2x80x9cacceptorxe2x80x9d). With regard to a fluorescent label and a quencher employed in a single-label detection procedure in an assay of the invention, it is additionally contemplated that the fluorophore and quencher are chosen such that the emission wavelength spectrum of the fluorophore is within the absorption spectrum of the quencher, such that when the fluorophore and the quencher with which it is employed are brought into close proximity by binding of the natural binding domain, sequence or polypeptide upon which one is present with the binding partner comprising the other, detection of the fluorescent signal emitted by the fluorophore is reduced by at least 10%, preferably 20-50%, more preferably 70-90% and, most preferably, by 95-100%. A typical quencher reduces detection of a fluorescent signal by approximately 80%.
According to one preferred embodiment, one of the isolated natural binding domain and the binding partner comprises a quencher for the detectable label.
The invention additionally provides a kit comprising an isolated natural binding domain and a binding partner therefor, wherein the isolated natural binding domain includes a site for post-translational modification and binds the binding partner in a manner dependent upon modification of the site, and packaging materials therefor.
It is preferred that the kit further comprises a buffer which permits modification-dependent binding of the isolated natural binding domain and the binding partner.
As used herein, the term xe2x80x9cbufferxe2x80x9d refers to a medium which permits activity of the protein-modifying enzyme used in an assay of the invention, and is typically a low-ionic-strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate. Such a buffer permits dimerization of a non-ubiquitinated and/or non-prenylated and/or non-sentrinated and/or non-ADP-ribosylated and/or non-glycosylated natural binding domain of the invention and a binding partner therefor and, preferably, inhibits degradation and maintains biological activity of the reaction components. Inhibitors of degradation, such as protease inhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease inhibitors (e.g., DEPC) are well known in the art. Lastly, an appropriate buffer may comprise a stabilizing substance such as glycerol, sucrose or polyethylene glycol.
As used herein, the term xe2x80x9cphysiological bufferxe2x80x9d refers to a liquid medium that mimics the salt balance and pH of the cytoplasm of a cell or of the extracellular milieu, such that post-translational protein modification reactions and protein:protein binding are permitted to occur in the buffer as they would in vivo.
Preferably, the buffer additionally permits modification of the site for protein modification by one or more of the following enzymes: a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme E1, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
It is preferred that the kit further comprises one or more of the following enzymes: carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme E1, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase.
It is additionally preferred that the kit further comprises a substrate for the enzyme which may be: ubiquitin, sentrin, nicotinamide adenine dinucleotide (NAD+), uridine-diphosphate-N-acetylglucosamine-dolichyl-phosphate (UDP-N-acetylglucosamine-dolichyl-phosphate), palmytyl CoA, myristoyl CoA and UDP-N-acetylglucosamine.
It is contemplated that at least a part of a substrate of an enzyme of use in the invention is transferred to an modification site on an isolated natural binding domain of the invention. As used herein, the term xe2x80x9cat least a part of a substratexe2x80x9d refers to a portion (e.g., a fragment of an amino acid sequence, a moiety or a group, as defined above) which comprises less than the whole of the substrate for the enzyme, the transfer of which portion to a modification site on an isolated natural binding domain and, optionally, to a site on a binding partner therefor, both as defined above, is catalyzed by the enzyme.
Preferably, the kit further comprises a cofactor for said enzyme.
It is preferred that at least one of the isolated natural binding domain and the binding partner comprises a detectable label, more preferred that the detectable label emits light and most preferred that the light is fluorescent.
An enzyme of use in the invention may be natural or recombinant or, alternatively, may be chemically synthesized. If either natural or recombinant, it may be substantially pure (i.e., present in a population of molecules in which it is at least 50% homogeneous), partially purified (i.e., represented by at least 1% of the molecules present in a fraction of a cellular lysate) or may be present in a crude biological sample.
As used herein, the term xe2x80x9csamplexe2x80x9d refers to a collection of inorganic, organic or biochemical molecules which is either found in nature (e.g., in a biological- or other specimen) or in an artificially-constructed grouping, such as agents which might be found and/or mixed in a laboratory. Such a sample may be either heterogeneous or homogeneous.
As used herein, the interchangeable terms xe2x80x9cbiological specimenxe2x80x9d and xe2x80x9cbiological samplexe2x80x9d refer to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). xe2x80x9cBiological samplexe2x80x9d and xe2x80x9cbiological specimenxe2x80x9d further refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. Lastly, xe2x80x9cbiological samplexe2x80x9d refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
As used herein, the term xe2x80x9corganismxe2x80x9d refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
In a method as described above, it is preferred that at least one of the isolated natural binding domain and the binding partner is labeled with a detectable label, more preferred that the label emits light and most preferred that the light is fluorescent.
Preferably, the detection step is to detect a change in signal emission by the detectable label.
According to one preferred embodiment, the method further comprises exciting the detectable label and monitoring fluorescence emission.
Preferably, the enzyme is one of the following enzymes: a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme E1, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase. The enzyme is not a protein kinase or phosphatase.
It is preferred that the method further comprises the step, prior to or after the detection step, of contacting the isolated natural binding domain and the binding partner with an agent which modulates the activity of the enzyme.
As used herein with regard to a biological or chemical agent, the term xe2x80x9cmodulatexe2x80x9d refers to enhancing or inhibiting the activity of a protein-modifying enzyme in an assay of the invention; such modulation may be direct (e.g. including, but not limited to, cleavage ofxe2x80x94or competitive binding of another substance to the enzyme) or indirect (e.g. by blocking the initial production or, if required, activation of the modifying enzyme).
xe2x80x9cModulationxe2x80x9d refers to the capacity to either increase or decease a measurable functional property of biological activity or process (e.g., enzyme activity or receptor binding) by at least 10%, 15%, 20%, 25%, 50%, 100% or more; such increase or decrease may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
The term xe2x80x9cmodulatorxe2x80x9d refers to a chemical compound (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule. Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein. The activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
The term xe2x80x9ccandidate modulatorxe2x80x9d refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.01 xcexcM, 0.1 xcexcM, 1.0 xcexcM, and 10.0 xcexcM, as described more fully hereinbelow. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
The invention also provides a method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme E1, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g, a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase, the method comprising contacting an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of the enzyme, wherein the natural binding domain includes a site for post-translational modification and binds the binding partner in a manner that is dependent upon modification of the site by the enzyme and wherein at least one of the isolated natural binding domain and the binding partner comprises a detectable label, and monitoring the binding of the isolated natural binding domain to the binding partner, wherein binding or dissociation of the isolated natural binding domain and the binding partner as a result of the contacting is indicative of modulation of enzymatic activity by the candidate modulator of the enzyme.
It is preferred that the detectable label emits light and highly preferred that the light is fluorescent.
Preferably, the monitoring comprises measuring a change in energy transfer between a label present on the isolated natural binding domain and a label present on the binding partner.
A final aspect of the invention is a method of screening for a candidate modulator of enzymatic activity of one or more of the following enzymes: a carbohydrate transferase (e.g., a UDP-N-Acetylglucosamine-Dolichyl-phosphate-N-acetylsglucosamine phosphotransferase or an O-GlcNAc transferase), a ubiquitin activating enzyme E1, a ubiquitin conjugating enzyme E2, a ubiquitin conjugating enzyme Ubc9, a ubiquitin protein ligase E3, a poly (ADP-ribose) polymerase, a fatty acyl transferase (e.g., a peptide N-myristoyltransferase) and an NAD:Arginine ADP ribosyltransferase, the method comprising contacting an assay system with a candidate modulator of enzymatic activity of such an enzyme, and monitoring binding of an isolated natural binding domain and a binding partner therefor in the assay system, wherein the natural binding domain includes a site for post-translational modification and binds the binding partner in a manner that is dependent upon modification of the site by at least one such enzyme in the assay system, wherein at least one of the isolated natural binding domain and the binding partner comprises a detectable label, and wherein binding or dissociation of the isolated natural binding domain and the binding partner as a result of the contacting is indicative of modulation of enzymatic activity by the candidate modulator of such an enzyme.
In a particularly preferred embodiment, in one of the methods described above, the method comprises real-time observation of association of an isolated natural binding domain and its binding partner.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.